Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (10): 2105-2117.doi: 10.3864/j.issn.0578-1752.2021.10.007

• PLANT PROTECTION • Previous Articles     Next Articles

Gene Cloning, Expression Pattern and Molecular Characterization of Chitin Deacetylase 2 in Cydia pomonella

SHI GuoLiang1,2(),WU Qiang2,3,YANG NianWan2,HUANG Cong3,LIU WanXue2,QIAN WanQiang3,WAN FangHao1,2,3()   

  1. 1College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao 266109, Shandong
    2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
    3Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences/Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs/Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen 518120, Guangdong
  • Received:2020-05-27 Accepted:2020-07-03 Online:2021-05-16 Published:2021-05-24
  • Contact: FangHao WAN E-mail:577389695@qq.com;wanfanghao@caas.cn

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Abstract:

【Objective】In order to provide a scientific basis for the selection of novel target for pest control, molecular characterization and expression patterns of chitin deacetylase 2 genes (CpCDA2) of the codling moth (Cydia pomonella) were studied. 【Method】The cDNA sequences of chitin deacetylase 2 genes were identified from the transcriptome data of C. pomonella by bioinformatic methods, and the conserved domain was analyzed, the homologous sequences from other insects were selected to construct phylogenetic tree by using neighbor-joining (NJ) method of MEGA7 software. The protein properties including molecular weight (Mw), theoretical isoelectric point (pI), glycosylation site and hydrophilic/hydrophobic properties were analyzed by online software. RT-qPCR was performed to detect the relative expression levels of CpCDA2a and CpCDA2b in different tissues and developmental stages. To explore the functions of these genes in the process of larval molting, the ecdysone (20E) was injected to the 5th instar larvae, and the expression dynamics of them were detected by RT-qPCR.【Result】The full length cDNA sequences of CpCDA2a and CpCDA2b, alternative splicing variants of C. pomonella CDA2, were identified. The amino acid analysis showed that both variants of CpCDA2 possess the signal peptide, and the open reading frames contain three conserved domains, including chitin binding domain (ChBD), low-density lipoprotein receptor class A domain (LDLa) and chitin deacetylase catalytic domain (CDA). The multi-sequence alignment analysis revealed that the CpCDA2a and CpCDA2b are similar to other insect, and the phylogenetic tree showed that the sequences of insect in the same order are clustered into a clade with high confidence. Three-dimensional structure simulation and comparison revealed that the ChBD functional domains of the two splicing variants are different in structure. Physical and chemical properties analysis showed that there are also differences in hydrophilic/hydrophobic properties and glycosylation site of the two splicing variants. Developmental expression patterns showed that the CpCDA2a expression was high in larval stage, CpCDA2b in early and middle stages of pupae, and the expression of CpCDA2a and CpCDA2b was significantly up-regulated before and after larval molting. Tissue-specific expression analysis indicated that the expression of CpCDA2a and CpCDA2b was the highest in integument, and the expression of CpCDA2b in head was also higher. Expression dynamics of the larvae after injection of ecdysone (20E) indicated that both CpCDA2a and CpCDA2b were up-regulated, but with very different levels.【Conclusion】The CpCDA2 has two alternatively spliced variants, namely CpCDA2a and CpCDA2b, which plays a key role in the development of C. pomonella. While, according to the comprehensive analysis of molecular characterization, developmental stages and tissue expression profiles, and expression trends after ecdysone injection, it is deduced that the differences in protein structure and physical and chemical properties of CpCDA2a and CpCDA2b caused by alternative splicing, resulted in the differentiation of function between them.

Key words: Cydia pomonella, chitin deacetylase 2, alternative splicing variant, molecular characterization, expression pattern, ecdysone, functional differentiation

Table 1

Information of primers used in this study"

基因名称 Gene name 引物序列 Primer sequence (5′-3′) 产物长度 Product length (bp) 用途 Application
CpCDA2a/CpCDA2b F: CAGTCCTCGGCGAGCGTTAT 1858/1841 cDNA克隆
cDNA cloning
R: CCACGCAAAAGGCATTCCAA
CpCDA2a qFc: TGTTGCTTGCTTGCTTCGTTAG 176 荧光定量PCR
RT-qPCR
qRa: GTTCTCGCCGCCCTTGTC
CpCDA2b qFc: TGTTGCTTGCTTGCTTCGTTAG 233
qRb: CCAGTCGCAGGTTTGTTTATCG
Cpβ-tubulin qF: GCGGGAACCAGATTGGAGCTAA 267
qR: ACTGGCCGAACACGAAGTTGTC

Fig. 1

Genome structure and protein domain analysis of CpCDA2a and CpCDA2b"

Fig. 2

Multiple alignment of CDA2 with partial amino acid sequences among different insects The black box indicates chitin binding domain (ChBD), green box indicates the 3rd exon (E3), red box indicates the 4th exon (E4)"

Fig. 3

Analysis of evolutionary relationship among CDA2 amino acid sequences of different insects"

Fig. 4

Three-dimensional structure of CpCDA2a and CpCDA2b proteins"

Table 2

Protein property of CpCDA2a and CpCDA2b"

蛋白性质
Protein property		 蛋白名称 Protein name
CpCDA2a CpCDA2b
氨基酸长度Amino acid length 542 aa 536 aa
分子量Molecular weight 61.58354 kD 60.94991 kD
等电点Theoretical pI 5.30 5.30
N-糖基化位点N-glycosylation N30, N296 N30, N290
O-糖基化位点O-glycosylation S19, T37*, T174, T273, S274, T383 S19, T168, T267, S268, T377

Fig. 5

Hydrophilic/hydrophobic analysis of CpCDA2a and CpCDA2b Gray boxes indicate the difference of CpCDA2a and CpCDA2b, the abscissa is the sequence position and the ordinate is the scale value of the amino acid (>0 means hydrophobicity, <0 means hydrophilicity)"

Fig. 6

The relative expressions of CpCDA2a and CpCDA2b at different developmental stages of C. pomonella β-tubulin is the reference, different letters on the bars mean significant difference of the same gene among different samples (P<0.05). The same as below"

Fig. 7

The relative expressions of CpCDA2a and CpCDA2b in different tissues of C. pomonella 4th instar larvae"

Fig. 8

Gene expression levels of CpCDA2 and CpCDA2b after 20E injection"

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